PROJECT SUMMARY The understanding of protein function is a primary obstacle to unlocking the basis of human health and disease. A powerful approach to unlocking the secrets of protein function is to reveal the intricacies of how an extremely diverse repertoire of proteins interact with each other. Conventional methods to screen for protein- protein interactions remain useful tools but suffer from critical limitations. In response to those limitations, proximity-dependent protein labeling approaches have been developed within the last several years. These methods are predominantly based on the cellular expression of proteins that are fused to enzymes that covalently biotinylate proximate proteins. These biotinylated proteins can subsequently be isolated and identified by mass spectrometry. One of these methods, called BioID, is based on the cellular expression of a promiscuous biotin ligase fused to a protein of interest. During a controlled labeling period the ligase will biotinylate proximate proteins to generate a history of protein-protein associations. Developed by our research group, BioID has become a widely used proximity-labeling approach as it is easy to employ, applicable to proteins that are refractory to study by conventional methods, and uniquely accumulates a record of protein associations that occurred during a defined labeling period to reveal low abundance and/or transient associations. Despite its current advantages there remain limitations to the BioID method and untested aspects of its capabilities that need to be addressed before the full potential of the method can be realized. Currently, the efficacy of promiscuous biotin ligases in the extracellular environment, secretory pathway or low pH organelles appears variably suboptimal. The practical labeling period for BioID currently prevents resolution of events that occur within a few hours or less. At temperatures below 37C BioID activity is reduced, interfering with effective application in several powerful model organisms. There is evidence that BioID can function in living organisms, which, if broadly applicable, would greatly expand the technique's potential. However there is no comprehensive analysis of BioID efficacy in the widely used mouse model. Also, it is unclear if BioID is capable of resolving discrete differences in protein behavior caused by subtle genetic mutations or changes in cellular conditions. Overall, research in the Roux laboratory seeks to evolve and broaden the capabilities of BioID to more effectively address a much broader array of biological questions. By taking advantage of the nuclear envelope biology expertise of the Roux lab and through established and new collaborations on more diverse topics, we will engage in methodology-driven studies designed to empower studies of protein function in important biological processes, to move beyond proof-of-principle studies and provide clear advances in scientific knowledge relevant to the understanding of human health and disease.